US9199102B2 - A3 adenosine receptor ligands for modulation of pigmentation - Google Patents

A3 adenosine receptor ligands for modulation of pigmentation Download PDF

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US9199102B2
US9199102B2 US13/386,339 US201013386339A US9199102B2 US 9199102 B2 US9199102 B2 US 9199102B2 US 201013386339 A US201013386339 A US 201013386339A US 9199102 B2 US9199102 B2 US 9199102B2
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adenosine receptor
adenosine
melanin
skin
meca
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US20120134945A1 (en
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Lea Levana Madi
Rafi Korenstein
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ORADIN PHARMACEUTICAL Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
    • A61K8/4926—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having six membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
    • A61K8/4933—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having sulfur as an exocyclic substituent, e.g. pyridinethione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/04—Preparations for care of the skin for chemically tanning the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/70—Biological properties of the composition as a whole

Definitions

  • the present invention relates to pharmaceutical and cosmetic compositions comprising A3 adenosine receptor ligands and methods of use thereof for modulating melanin production, secretion and/or accumulation in skin.
  • a variety of dermatological compositions have been suggested for skin whitening to counteract abnormal pigmentation occurring in various disorders.
  • Such disorders include post inflammatory hyperpigmentation, (attributed to various preceding conditions such as infections, allergic reactions, mechanical injuries, reactions to medications, phototoxic eruptions, trauma for example burns, and inflammatory diseases such as lichen planus, lupus erythematosus, atopic dermatitis), melasma, periorbital darkening, pigmented keratosis, lentigo senilis, ephelides, chloasma, café au lait spots, liver spots, freckles and lesions observed in Addison's disease, hemochomatosis, piebaldism, and the like.
  • melanosomes In mammals, skin and hair color is primarily determined by the amount of melanin pigments that are synthesized by melanocytes within specialized organelles called melanosomes. There is no evidence for differences in melanosome biogenesis between follicular and epidermal melanocytes. Thus, in black hair follicles, melanocytes contain the largest number and most electron-dense melanosomes. In brown hair, bulb melanocytes are smaller, and in blonde hair melanosomes are poorly melanized (Slominski et al., 2004).
  • Melanin synthesis (also termed melanogenesis) is controlled by at least three enzymes: tyrosinase, tyrosinase-related-protein 1 (TRP1) and tyrosinase-related-protein 2 (TRP2), among which tyrosinase has the key role of catalyzing the rate limiting steps of the hydroxylation reaction of tyrosine to L-DOPA and the oxidation reaction of L-DOPA into dopaquinone.
  • TRP1 tyrosinase-related-protein 1
  • TRP2 tyrosinase-related-protein 2
  • ⁇ -MSH keratinocyte-derived melanocyte stimulating hormone
  • Keratinocyte growth factor a paracrine mediator of human keratinocyte growth and differentiation, has been shown to induce melanosome transfer from melanocytes to keratinocytes through a phagocytic process (Cardinali et al., 2005).
  • Dermatological compositions which have been disclosed for hair and skin bleaching typically act by destroying or disrupting melanin granules, inhibiting melanin formation (such as by inhibiting tyrosinase or melanocyte activity), or both.
  • Various skin whitening compositions are disclosed for example, in U.S. Pat. Nos. 5,980,904, 5,747,006, and 6,077,503. Many of these compositions contain harsh chemicals such as peroxides, acids or formaldehyde, or thiolated compounds such as glutathione, cysteine, mercaptosuccinic acid, mercaptodextran, and mercaptoethanol, which have an objectionable odor that makes products containing them undesirable for use.
  • Hydroquinone for topical application is approved in the United States for non-prescription use, and acts by suppressing melanocyte activity.
  • U.S. Pat. No. 6,068,834 discloses hydroquinone-based compounds and compositions Hydroquinone, however, is oxidized by air, light, and tyrosinase itself, which adversely affects the shelf life of preparations containing it and its bioavailability upon application. Hydroquinone can cause burning, redness, sensitization and irritation in some individuals, particularly after application of quantities sufficient to cause skin bleaching. Oxidized products of hydroquinone have also been implicated in skin irritation and pigmentation rebound.
  • Retinoids and corticosteroids for topical use have been suggested as hypopigmenting agents, as have laser treatment and chemical peels, but these fall short of desirable responses.
  • a combination therapy containing tretinoin and fluocinolone with hydroquinone has been disclosed (Willis, 2000).
  • Kojic acid and arbutin have also been suggested (Draelos, 2007), but these weak tyrosinase inhibitors generally display low bioavailability.
  • arbutin is ineffective at levels allowed by Japanese quasi-drug regulations, and kojic acid has been banned for quasi-drug usage in Japan due to its mutagenic properties (Boissy et al., 2005).
  • U.S. Pat. No. 7,019,029 discloses the use of hydroxythtronic acid derivatives alone or in combination with tetronic acid, hydroquinone, glycolic acid and/or ascorbyl palmitate as skin whiteners.
  • U.S. Pat. No. 5,905,091 discloses a composition comprising a carrier and a prostaglandin, and optionally comprising a lysosomotropic agent, a phosphodiesterase inhibitor, and/or methylxanthines, and a method of use thereof for stimulating synthesis of melanin in a human melanocyte thereby enhancing pigmentation of human skin.
  • U.S. Pat. No. 5,554,359 discloses a composition comprising a lysosomotropic agent, and optionally phosphodiesterase inhibitors, and/or methylxanthines for increasing synthesis of melanin in a human melanocyte thereby enhancing pigmentation of human skin.
  • Adenosine is an endogenous purine nucleoside ubiquitous in mammalian cells, which is an important regulatory compound that mediates many physiological effects via binding to its specific A1, A2 and A3 cell surface receptors. Interaction of adenosine with its receptors initiates signal transduction pathways, in particular that of the adenylate cyclase effector system, which utilizes cAMP as a second messenger.
  • the A1 and A3 adenosine receptors which are coupled to Gi proteins, inhibit adenylate cyclase and lead to a decrease in the level of intracellular cAMP, while the A2 adenosine receptor, which is coupled to Gs proteins, activates adenylate cyclase, thereby increasing cAMP levels (see for example, Fredholm et al., 2000).
  • adenosine receptor agonists and antagonists for treatment of different disease states and pathologies has been suggested, including for example, inflammation (Salvatore et al., 2000), neurodegeneration (Von Lubitz, 1999), asthma (Forsythe and Ennis, 1999), cardiac ischemia (Liang and Jacobson, 1998), and tumors (Yao et al., 1997).
  • U.S. Pat. No. 7,064,112 discloses the use of A3 adenosine receptor agonists for the prevention and treatment of leukopenia, and for the inhibition of abnormal cell growth and proliferation of tumor cells.
  • WO 2008/023362 discloses methods and compositions useful for treating cancer based on the combination of methotrexate and an A3 adenosine receptor agonist.
  • U.S. Pat. No. 7,465,715 discloses the use of A3 adenosine receptor agonists for the treatment of multiple sclerosis.
  • U.S. Pat. No. 7,465,715 discloses the use of A3 adenosine receptor agonists for inhibiting viral replication in cells.
  • U.S. Pat. No. 7,141,553 discloses the use of A3 adenosine receptor agonists for the treatment of inflammatory arthritis.
  • U.S. Pat. No. 5,998,423 discloses the use of adenosine A1 receptor antagonists and of adenosine A2 receptor agonists for increasing melanin production in skin or hair, and the use of adenosine A1 receptor agonists and adenosine A2 receptor antagonists for decreasing melanin production in skin or hair.
  • U.S. Patent Application Publication No. 2002/0115635 discloses a treatment method comprising administering to a subject an active agent selected from the group consisting of an adenosine A1 receptor ligand, an A2 adenosine receptor ligand, an adenosine A3 receptor ligand and a combination thereof.
  • the method is disclosed to be effective for a variety of diseases and disorders, inter alia hair loss, which require elevation of GSK-3 ⁇ activity for treatment.
  • U.S. Patent Application Publication No. 2007/0299032 discloses a method for maintaining and promoting hair thickening comprising applying to the scalp an external skin preparation containing one or more types of agents, inter alia the A3 adenosine receptor agonist Cl-IB-MECA.
  • U.S. Patent Application Publication No. 2008/0044439 discloses use of p38 inhibitors for treating skin conditions such as vitiligo, and describes adenosine A3 antagonists as possible p38 inhibitors.
  • the present invention provides methods as well as pharmaceutical and cosmetic compositions useful for modulating the production, secretion and/or accumulation of melanin in skin, in particular within skin cells such as melanocytes and keratinocytes, thereby modulating pigmentation of the skin and hair.
  • the present invention provides use of A3 adenosine receptor antagonists in compositions and methods for the treatment and amelioration of hyper-pigmentation skin conditions and for cosmetic lightening of skin and hair color, as well as use of A3 adenosine receptor agonists in compositions and methods for the treatment and amelioration of hypo-pigmentation skin conditions and for cosmetic skin tanning.
  • the present invention is based in part on the surprising discovery that activation of the A3 adenosine receptor causes an increase in pigmentation, while deactivation of the A3 adenosine receptor causes a decrease in pigmentation.
  • the present invention is highly unexpected over the prior art, which teaches that activation of the A3 adenosine receptor, which is known to be negatively coupled to adenylate cyclase, should inhibit melanogenesis due to decreased intracellular levels of cAMP and thus lead to depigmentation.
  • melanocytes treated with the A3 adenosine receptor agonist IB-MECA unexpectedly exhibit an increase in pigmentation, attributable to an increase in melanin secretion by the melanocytes.
  • the inventors of the present invention have shown that treatment of melanoctyes with the A3 adenosine receptor antagonist MRS-1523, unexpectedly led to depigmentation.
  • A3 adenosine receptor ligands may exert modulation of pigmentation by altering melanin production and/or secretion in melanocytes, as well as altering melanin accumulation in keratinocytes and/or melanin secretion from melanocytes to keratinocytes.
  • the effect of A3 adenosine receptor agonists in enhancing pigmentation and the effect of A3 adenosine receptor antagonists in decreasing pigmentation may respectively involve Mitf activation and Mitf downregulation, and mediation thereof via the Akt and ERK signaling pathways.
  • the present invention provides a method for modulating melanin production, secretion, accumulation or a combination thereof in at least one skin cell, comprising the step of contacting the skin cell with a compound selected from the group consisting of an A3 adenosine receptor antagonist and an A3 adenosine receptor agonist in an amount effective to modulate melanin production, secretion, and/or accumulation in the skin cell.
  • the at least one skin cell is a melanocyte, a keratinocyte, or tissue comprising a plurality of melanocytes and/or keratinocytes.
  • the compound is an A3 adenosine receptor agonist
  • the modulating comprises increasing at least one of melanin production, secretion or accumulation.
  • the skin cell is a melanocyte
  • the A3 adenosine receptor agonist is in an amount effective to increase at least one of melanin production and melanin secretion in said melanocyte.
  • the skin cell is a keratinocyte
  • the A3 adenosine receptor agonist is in an amount effective to increase accumulation of melanin in said keratinocyte.
  • the A3 adenosine receptor agonist is in an amount effective to increase secretion of melanin from a melanocyte to a keratinocyte.
  • the A3 adenosine receptor agonist has a binding affinity (Ki) for the A3 adenosine receptor that is less than about 200 nM, preferably less than about 100 nM, more preferably less than about 50 nM, even more preferably less than about 10 nM.
  • Ki binding affinity
  • the binding affinity of the A3 adenosine receptor agonist for the adenosine A3 receptor is at least 20 times greater than the binding affinity of said agonist for the A1 adenosine receptor.
  • the A3 adenosine receptor is the human A3 adenosine receptor.
  • the A3 adenosine receptor agonist is a selective A3 adenosine receptor agonist.
  • the A3 adenosine receptor agonist is an N 6 -substituted-adenosine-5′-uronamide.
  • the N 6 -substituted-adenosine-5′-uronamide is selected from the group consisting of an N 6 -monosubstituted-adenosine-5′-uronamide and an N 6 -disubstituted-adenosine-5′-uronamide.
  • the A3 adenosine receptor agonist is selected from the group consisting of an N 6 -benzyladenosine-5′-uronamide; an N 6 -4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide; a 2-chloro-N 6 -substituted-4′-thioadenosine-5′-uronamide; an (N)-methanocarba adenosine-5′-uronamide and derivatives and analogs thereof.
  • N 6 -benzyladenosine-5′-uronamide an N 6 -4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide
  • 2-chloro-N 6 -substituted-4′-thioadenosine-5′-uronamide an (N)-methanocarba adenosine-5′-uronamide and derivative
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA); 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA); N 6 -(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (AB-MECA); N 6 -2-(4-aminophenyl) ethyladenosine (APNEA); 2-(1-hexynyl)-N-methyladenosine, 2-chloro-N 6 -(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine (CF-102
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA); 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA); N 6 -(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (AB-MECA); and N 6 -2-(4-aminophenyl) ethyladeno sine (APNEA).
  • IB-MECA 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)
  • Cl-IB-MECA 2-chloro-N 6 -(3-iodobenzyl)-aden
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA); 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA); 2-(1-hexynylhexynyl)-N-methyladenosine; 2-chloro-N 6 -(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine (CF-102); and (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methyl
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA) and 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA).
  • the A3 adenosine receptor agonist is N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA).
  • the method for increasing at least one of melanin production, secretion or accumulation further comprises contacting the skin cell with an allosteric modulator of the A3 adenosine receptor.
  • the allosteric modulator is an allosteric enhancer of the A3 adenosine receptor.
  • the skin cell is in a human subject having a hypo-pigmentation disorder selected from the group consisting of vitiligo, piebaldism, leukoderma due to cicatrisation, nevus depigmentosis and depigmentation due to a skin graft procedure.
  • a hypo-pigmentation disorder selected from the group consisting of vitiligo, piebaldism, leukoderma due to cicatrisation, nevus depigmentosis and depigmentation due to a skin graft procedure.
  • the compound is an A3 adenosine receptor antagonist
  • the modulating comprises decreasing at least one of melanin production, secretion or accumulation in the skin cell.
  • the skin cell is a melanocyte
  • the A3 adenosine receptor antagonist is in an amount effective to decrease at least one of melanin production and melanin secretion in said melanocyte.
  • the skin cell is a keratinocyte
  • the A3 adenosine receptor antagonist is in an amount effective to decrease accumulation of melanin in said keratinocyte.
  • the A3 adenosine receptor antagonist is in an amount effective to decrease secretion of melanin from a melanocyte to a keratinocyte.
  • the A3 adenosine receptor antagonist has a binding affinity (Ki) for the A3 adenosine receptor that is less than about 200 nM, preferably less than about 100 nM, more preferably less than about 50 nM, even more preferably less than 10 nM.
  • Ki binding affinity
  • the binding affinity of the A3 adenosine receptor antagonist for the adenosine A3 receptor is at least 20 times greater than the binding affinity of said antagonist for the A1 adenosine receptor.
  • the A3 adenosine receptor is the human A3 adenosine receptor.
  • the A3 adenosine receptor antagonist is a selective A3 adenosine receptor antagonist.
  • the A3 adenosine receptor antagonist is a compound selected from the group consisting of a dihydropyridine, a pyridine, a pyridinium salt, a triazoloquinazoline, an imidazoquinoline, a triazolopurine, an N 6 -substituted-7-deazapurine, and derivatives and analogs thereof.
  • a dihydropyridine a pyridine
  • a pyridinium salt a triazoloquinazoline
  • an imidazoquinoline a triazolopurine
  • an N 6 -substituted-7-deazapurine and derivatives and analogs thereof.
  • the A3 adenosine receptor antagonist is selected from the group consisting of 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523); 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334); 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-( ⁇ )-dihydropyridine-3,5 dicarboxylate (MRS-1191); 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-( ⁇ )-dihydropyridine-3,5-dicarboxylate (MRS-1097); 5-n-butyl-8-(4-methyl-6-phen
  • the A3 adenosine receptor antagonist is selected from the group consisting of 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523); 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyemethyl] ester (MRS-1334); and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-( ⁇ )-dihydropyridine-3,5 dicarboxylate (MRS-1191).
  • the A3 adenosine receptor antagonist is 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523).
  • the method further comprises the step of contacting the skin cell with at least one of hydroxytetronic acid; tetronic acid; hydroquinone; an ⁇ -hydroxy acid, a fatty acid ester of ascorbic acid; a tyrosinase inhibitor and a tyrosine phosphatase inhibitor.
  • the skin cell is in a human subject having a hyper-pigmentation disorder selected from the group consisting of pigmented spots, lentigo senilis, freckles, café au lait spots, liver spots, ephelides, periorbital darkening, post-inflammatory hyper-pigmentation, pigmented keratosis, melasma, chloasma, and hyper-pigmentation due to a skin graft procedure.
  • a hyper-pigmentation disorder selected from the group consisting of pigmented spots, lentigo senilis, freckles, café au lait spots, liver spots, ephelides, periorbital darkening, post-inflammatory hyper-pigmentation, pigmented keratosis, melasma, chloasma, and hyper-pigmentation due to a skin graft procedure.
  • the invention further provides a compound selected from the group consisting of an A3 adenosine receptor antagonist and an A3 adenosine receptor agonist for use in modulating melanin production, secretion, accumulation or a combination thereof in at least one skin cell.
  • the compound is an A3 adenosine receptor agonist in an amount effective for increasing at least one of melanin production, secretion or accumulation. In another particular embodiment, the compound is an A3 adenosine receptor antagonist in an amount effective for decreasing at least one of melanin production, secretion or accumulation.
  • the present invention provides a cosmetic or pharmaceutical composition for use in modulating at least one of melanin production, secretion or accumulation in skin, wherein the composition comprises as an active ingredient an A3 adenosine receptor antagonist or an A3 adenosine receptor agonist; and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is capable of delivering the active ingredient to a skin cell under in vivo conditions.
  • the composition comprises an A3 adenosine receptor agonist as described herein, in an amount effective for increasing at least one of melanin production, secretion or accumulation in skin.
  • an A3 adenosine receptor agonist as described herein, in an amount effective for increasing at least one of melanin production, secretion or accumulation in skin.
  • the composition comprises an A3 adenosine receptor agonist in an amount effective for increasing at least one of melanin production and melanin secretion in a melanocyte.
  • the composition comprises an A3 adenosine receptor agonist in an amount effective for increasing melanin accumulation in a keratinocyte.
  • the composition comprises an A3 adenosine receptor agonist in an amount effective for increasing melanin secretion from a melanocyte to a keratinocyte.
  • the composition comprises N 6 -(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA) as the A3 adenosine receptor agonist.
  • composition comprising an A3 adenosine receptor agonist, further comprises an allosteric enhancer of the A3 adenosine receptor.
  • the composition comprises an A3 adenosine receptor antagonist as described herein, in an amount effective for decreasing at least one of melanin production, secretion or accumulation in skin.
  • an A3 adenosine receptor antagonist as described herein, in an amount effective for decreasing at least one of melanin production, secretion or accumulation in skin.
  • the composition comprises an A3 adenosine receptor antagonist in an amount effective for decreasing at least one of melanin production and melanin secretion in a melanocyte.
  • the composition comprises an A3 adenosine receptor antagonist in an amount effective for decreasing melanin accumulation in a keratinocyte.
  • the composition comprises an A3 adenosine receptor antagonist in an amount effective for decreasing melanin secretion from a melanocyte to a keratinocyte.
  • the composition comprises 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523) as the A3 adenosine receptor antagonist.
  • the composition comprising an A3 adenosine receptor antagonist further comprises at least one of hydroxytetronic acid; tetronic acid; hydroquinone; an ⁇ -hydroxy acid, a fatty acid ester of ascorbic acid; a tyrosinase inhibitor and a tyrosine phosphatase inhibitor.
  • compositions are formulated for administration by a route selected from the group consisting of direct administration into, onto or in the vicinity of a skin site of hyper-pigmented or hypo-pigmented tissue, topical, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral administration.
  • the present invention provides a method for treating or ameliorating a hyper-pigmentation condition of human skin, the method comprising administering to a human subject in need thereof an effective amount of a cosmetic or pharmaceutical composition comprising as an active ingredient an A3 adenosine receptor antagonist according to the principles of the invention.
  • the hyper-pigmentation condition is selected from the group consisting of pigmented spots, lentigo senilis, freckles, café au lait spots, liver spots, ephelides, periorbital darkening, post-inflammatory hyper-pigmentation, pigmented keratosis, melasma, chloasma and hyper-pigmentation due to a skin graft procedure.
  • the composition is a cosmetic composition for lightening dark skin.
  • a cosmetic or pharmaceutical composition comprising an A3 adenosine receptor antagonist for use in treating or ameliorating a hyper-pigmentation condition of human skin, in accordance with the invention.
  • the present invention provides a method for treating or ameliorating a hypo-pigmentation condition of human skin comprising administrating to a human subject in need thereof an effective amount of a cosmetic or pharmaceutical composition comprising as an active ingredient an A3 adenosine receptor agonist according to the principles of the invention.
  • the hypo-pigmentation condition is selected from the group consisting of pigmented vitiligo, piebaldism, leukoderma due to cicatrisation, nevus depigmentosis, and depigmentation due to a skin graft procedure.
  • the composition is a cosmetic composition for tanning fair skin.
  • the administering comprises topically applying the composition comprising the A3 adenosine receptor agonist or the A3 adenosine receptor antagonist to skin of the subject.
  • the A3 adenosine receptor agonist or the A3 adenosine receptor antagonist is topically administered over a period of at least two weeks, or at least one month, or at least two months.
  • a cosmetic or pharmaceutical composition comprising an A3 adenosine receptor agonist for use in treating or ameliorating a hypo-pigmentation condition of human skin, in accordance with the invention.
  • the administering of the cosmetic or pharmaceutical composition to the subject is carried out by a route selected from the group consisting of direct administration into, onto or in the vicinity of hyper-pigmented or hypo-pigmented skin, topical, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral administration.
  • the composition is topically administered.
  • the cosmetic or pharmaceutical composition is in a form selected from the group consisting of a solution, a suspension, an emulsion, a cream, a gel, an aerosol formulation and a sustained-release formulation.
  • FIG. 1 shows the effect of the A3 adenosine receptor agonist IB-MECA on melanin synthesis and secretion in B16 melanocytes.
  • Cells were cultured in 10% DMEM in the presence of various concentrations of IB-MECA for 5 days.
  • Levels of melanin in the media and in the cell lysates, and total protein content in the cell lysates were then determined as described in Materials and Methods. Melanin levels were measured and calculated separately for each sample.
  • FIG. 1A shows melanin levels in cell lysates and in the media expressed as ⁇ g melanin/mg protein.
  • FIG. 1C is a photograph showing the dose response of melanin secretion to the media by cells treated with the indicated concentrations of IB-MECA.
  • FIG. 2 shows the effect of the A3 adenosine receptor antagonist MRS-1523 (abbreviated as “MRS”) on melanin synthesis and secretion in B16 melanocytes.
  • MRS-1523 A3 adenosine receptor antagonist MRS-1523
  • Cells were cultured in 10% DMEM in the presence of various concentrations of MRS-1523 for 5 days.
  • Levels of melanin in the media and in the cell lysates, and total protein content in the cell lysates were then determined as described in Materials and Methods. Melanin levels were measured and calculated separately for each sample.
  • FIG. 2A shows melanin levels in cell lysates and in the media expressed as ⁇ g melanin/mg protein.
  • FIG. 3 shows the time course of melanin synthesis and secretion by B16 melanocytes treated with IB-MECA or MRS-1523.
  • Cells were exposed to 10 ⁇ M of IB-MECA or MRS-1523 and the melanin was monitored at different time intervals.
  • Levels of melanin in the media and in the cell lysates, and total protein content in the cell lysates were determined as described in Materials and Methods. Melanin levels were measured and calculated separately for each sample.
  • FIG. 3A is a photograph of the cells following treatment with the indicated compounds for 24 h. Arrows indicate positions of melanosomes. N, nucleus.
  • FIG. 3B depicts transfer of melanosomes from one melanocyte to the other by a dendrite-dendrite interaction. Arrow indicates position of melanosomes.
  • FIG. 3C shows total melanin levels after various treatment intervals expressed as percent of control.
  • FIG. 3D shows melanin levels expressed as ⁇ g melanin/mg protein in cell lysates and in the media following exposure of melanocytes to IB-MECA or MRS-1523 for 5-days.
  • FIG. 3E is a photograph showing melanin secretion to the media following 5 days of exposure to IB-MECA or MRS-1523, as indicated.
  • FIG. 4 shows the effect of kojic acid on melanin synthesis and secretion.
  • B16 melanocytes were cultured in 10% DMEM in the presence of Kojic acid for 5 days.
  • Levels of melanin in the media and in the cell lysates, and total protein content in the cell lysates were determined as described in Materials and Methods. Melanin levels were measured and calculated separately for each sample.
  • FIG. 4A shows melanin levels in cell lysates and in the media expressed as ⁇ g melanin/mg protein.
  • FIG. 5 shows the effect of A3 adenosine receptor ligands on Akt phosphorylation. Serum starved B16 melanocytes were exposed to 10 ⁇ M IB-MECA or MRS-1523 for 10 min, 30 min or 3 h.
  • FIG. 5A shows western blot analysis of cell protein extracts (20 ⁇ g/lane) analyzed for Akt phosphorylation compared to total Akt expression. Time intervals of exposure to the ligands are indicated.
  • FIG. 5B shows confocal image analysis of B16 melanocytes exposed to the indicated ligands for 3 h, following labeling with anti-phospho-Akt antibody and Cy3-conjugated donkey anti-rabbit antibody, and counter staining with DAPI. Arrow indicates phospho-Akt. Scale bars correspond to 25 ⁇ .
  • FIG. 6 shows the effect of A3 adenosine receptor ligands on ERK1/2 phosphorylation.
  • Serum starved B16 melanocytes were exposed to 10 ⁇ M IB-MECA or MRS-1523 for various time periods, and protein extracts of the cells were analyzed by western blot analysis (20 ⁇ g protein/lane) for ERK phosphorylation and total ERK expression.
  • FIG. 6A shows analysis of proteins from cells exposed to the indicated ligands for 10 min, 30 min or 3 h.
  • FIG. 6B shows analysis of proteins from cells exposed to the indicated ligands for 72 h.
  • FIG. 7 shows Mitf expression and localization in B16 melanocytes treated with A3 adenosine receptor ligands for 3 h or 24 h, followed by immunostaining with anti-Mitf antibody and visualization by confocal microscopy.
  • N nucleus.
  • FIG. 8 shows the effect of A3 adenosine receptor ligands on skin pigmentation as evaluated by DOPA staining.
  • Samples of human breast skin were topically treated with A3 adenosine receptor ligands (10 or 50 ⁇ M) or with hydroquinone (100 ⁇ M) as indicated three times over a culture period of 12 days, during which the medium was replaced every 3 days.
  • FIG. 8A is a photograph of skin samples following treatment with the indicated compounds after 12 days of culture.
  • FIG. 8B shows light microscope images ( ⁇ 400) of epidermis peeled from skin samples treated with IB-MECA or MRS-1523 (10 ⁇ M each) or hydroquinone (100 ⁇ M) as indicated, stained with DOPA and mounted on slides.
  • FIG. 8D shows light microscope images ( ⁇ 400) of epidermis peeled from skin samples treated with IB-MECA or MRS-1523 (50 ⁇ M each) or hydroquinone (100 ⁇ M) as indicated, stained with DOPA and mounted on slides.
  • FIGS. 8C and 8E show the degree of pigmentation (density) in the samples shown in FIGS. 8B and 8D respectively, as evaluated by measurement of the light transmission capacity though the peeled epidermis sheets using single channel transmission by light microscopy ( ⁇ 50).
  • D-P-M DOPA-positive melanocytes.
  • FIG. 9 shows the effect of A3 adenosine receptor ligands on skin pigmentation as evaluated by Fontana-Masson stain.
  • Formalin fixed paraffin embedded skin sections from samples of human breast skin treated with IB-MECA, MRS-1523 or hydroquinone as described for FIG. 8 were evaluated for Fontana-Masson staining and visualized by light microscopy ( ⁇ 400).
  • IBMX 100 ⁇ M was used as a positive control for induction of melanogenesis.
  • K keratinocytes
  • M melanocytes.
  • the present invention provides methods as well as pharmaceutical and cosmetic compositions useful for modulating i.e. increasing or decreasing, the production, secretion and/or accumulation of melanin in skin cells, thereby enabling alteration of pigmentation of the skin and hair.
  • the present invention provides methods and compositions comprising use of A3 adenosine receptor agonists for enhancing the tanning process by increasing skin pigmentation, as well as methods and compositions comprising use of A3 adenosine receptor antagonists for reducing skin pigmentation to lighten the skin color.
  • A3 adenosine receptor agonists such as IB-MECA
  • A3 adenosine receptor agonists cause an increase in melanin synthesis in melanocytes in a dose-dependent fashion, as shown in Example 1.
  • A3 adenosine receptor agonists cause a significant increase in melanin secretion from the cells, as shown in Example 3.
  • agonist treatment further exerts the effect of altering accumulation and/or re-distribution of melanosomes within the cell, as shown in Example 3, and moreover may promote melanosome transfer from melanocytes to keratinoctyes, as shown in Example 9.
  • Example 3 further shows that the increase in melanin production occurs in a time-dependent fashion, indicating that the invention may be effectively used for gradually increasing skin pigmentation, which is advantageous both for cosmetic applications directed at achieving “tanning” effects over a period of time, and for pharmaceutical applications directed at ameliorating hypo-pigmentation skin disorders.
  • A3 adenosine receptor antagonists such as MRS-1523, cause a decrease in melanin synthesis in melanocytes in a dose dependent fashion, as shown in Example 2. This reduction also occurs in a time-dependent fashion, as shown in Example 3.
  • the principles of the invention directed to reducing skin pigmentation have further been demonstrated in ex vivo studies of skin explants as shown in Examples 8 and 9, which provide confirmation of the decrease in melanin production and/or secretion in melanocytes and/or melanin accumulation in keratinocytes using a number of histological and microscopic methods.
  • Examples 5 to 7 suggest that the effect of A3 adenosine receptor agonists in enhancing pigmentation, and the effect of A3 adenosine receptor antagonists in decreasing pigmentation, may involve Mitf activation and Mitf downregulation respectively, mediation of which occurs via the Akt and ERK signaling pathways.
  • the term “ligand of the A3 adenosine receptor” refers to a compound which specifically binds the A3 adenosine receptor and thereby causes either activation of the A3 adenosine receptor (i.e., A3 adenosine receptor agonist), or inhibition of the A3 adenosine receptor (i.e., A3 adenosine receptor antagonist).
  • A3 adenosine receptor agonist refers to a ligand of the A3 adenosine receptor, which upon binding to the receptor exerts full or partial activation of that receptor.
  • A3 adenosine receptor antagonist refers to a ligand of the A3 adenosine receptor, which upon binding to the receptor exerts full or partial inhibition of that receptor.
  • the term “selective” means that the binding affinity of an A3 adenosine receptor agonist or of an A3 adenosine receptor antagonist to the A3 adenosine receptor is at least 20 fold, preferably at least 50 fold and more preferably at least 100 fold greater than the binding of the same compound to a heterologous adenosine receptor, for example the A1 adenosine receptor, the A2a adenosine receptor or the A2b adenosine receptor of the same species, or the A3 adenosine receptor of a different species.
  • allosteric enhancer of the A3 adenosine receptor refers to a compound which enhances the binding of an A3 adenosine receptor agonist to the A3 adenosine receptor, thereby increasing the activation effect of the agonist.
  • modulating melanin production means affecting a change in the pattern of melanin production, for example increasing or decreasing the overall amount of melanin produced by a cell, or increasing or decreasing the rate of melanin production by a cell.
  • the term “modulating melanin secretion” means affecting a change in the pattern of melanin secreted from a cell, for example increasing or decreasing the overall amount of melanin secreted from a cell, or increasing or decreasing the rate of melanin secreted from a cell.
  • the term also encompasses an alteration in the target destination of melanin secretion such as into the extracellular medium or space, or into a different cell or into a different cell type.
  • modulating melanin accumulation means affecting a change in the pattern of melanin deposition or sequestration in or among cell compartments or organelles, or among cell types.
  • skin cell refers to any type of skin cell, including skin cells involved in melanin production and/or accumulation such as melanocytes and keratinocytes, and precursors thereof.
  • the term “at least one skin cell” encompasses both a single skin cell and a plurality of skin cells, including those forming skin tissue and skin surface areas of any size or dimension, for example as measured in square meters of body surface area.
  • an A3 adenosine receptor agonist is any compound capable of specifically binding to the A3 adenosine receptor (also referred to herein as “A3R”), and capable of fully or partially activating said receptor.
  • A3RAg is thus a compound that exerts its prime effect though the binding and activation of the A3R.
  • the ability of a compound to bind A3R may be assessed in a competitive binding assay, typically in which a test compound is assessed for the ability to displace a radiolabeled form of a known A3RAg (for example [125I]-AB-MECA) from binding to the A3R present on cells or membranes.
  • binding assays are described for example in Olah et al., Mol Pharmacol. 1994 May; 45(5):978-82; Auchambach et al. Mol Pharmacol. 1997 November; 52(5):846-60; and Kreckler et al., J Pharmacol Exp Ther. 2006 April; 317(1):172-80.
  • the ability of a compound to activate A3R may also be assessed in a functional assay based on determination of downstream signaling events, in particular the effect on adenylyl cyclase as measured by the effect (i.e. increase or decrease) on cAMP level.
  • Such cAMP assays used to assess A3R activation by various compounds are described for example in Wan et al., J Pharmacol Exp Ther. 2008 January; 324(1):234-43 and Auchambach et al. Mol Pharmacol. 1997 November; 52(5): 846-60.
  • the affinity of an A3RAg to the human A3R as well as its relative affinity to the other human adenosine receptors can be determined by binding assays and cAMP assays, as described above.
  • the A3RAg is a selective A3RAg, meaning that its binding affinity for the A3 adenosine receptor is greater than the binding affinity of the same compound for a different adenosine receptor, for example the A1 adenosine receptor.
  • the binding affinity of the A3RAg for the A3 adenosine receptor is at least 20 times greater than the binding affinity of said agonist for the A1 adenosine receptor.
  • an A3RAg has a binding affinity (K i ) for the human A3 adenosine receptor in the range of less than 200 nM, typically less than 100 nM, preferably less than 50 nM, more preferably less than 20 nM and even more preferably less than 10 nM.
  • Ki the lower the Ki, the lower the dose of the A3RAg that may be used that will be effective in activating the A3R and thus achieving the desired effect.
  • an A3RAg that has a K i for the human A3R of less than 5 nM and even less than 1 nM may be preferred.
  • the A3RAg specifically activates the A3R at the administered levels, and is substantially devoid of activity in activating any of the other adenosine receptors i.e. the A1, A2a and A2b adenosine receptors.
  • the administered level of an A3RAg is such that its blood level reaches a level approaching that of the K i of the A1, A2a or A2b adenosine receptors, activation of these receptors may occur following such administration, in addition to activation of the A3R.
  • An A3RAg is thus preferably administered at a dose such that the blood level that will be attained will give rise to essentially only A3R activation.
  • A3 adenosine receptor agonists which are useful in the present invention include a variety of nucleoside derivatives which are known to act as A3 adenosine receptor agonists.
  • nucleoside refers to a compound comprising a sugar backbone, preferably ribose or deoxyribose, linked to a purine or pyrimidine base by way of N-glycosyl link.
  • nucleoside-derivative denotes herein a synthetic nucleoside or a nucleoside which underwent chemical modifications by way of insertion/s, deletion/s or exocyclic and endocyclic substitution/s of group/s therein or conformational modifications which provide a derivative with the desired biological effect, such as enhancing melanin production and/or secretion, thereby increasing pigmentation in skin.
  • the desired biological effect caused by an A3 adenosine receptor agonist is enhancement of melanin synthesis and/or secretion and/or accumulation in a skin cell.
  • A3 adenosine receptor agonists useful for the invention include N 6 -benzyladenosine-5′-uronamide derivatives, especially those containing a methyl- or ethyl-substituted uronamide moiety, which have been shown to possess significant A3 adenosine receptor affinity and selectivity.
  • a well known A3 adenosine receptor agonist is N 6 -(3-iodo-benzyl)-adenosine-5′-N-methyl-uronamide (IB-MECA) which is known to be 50-fold selective for the A3 adenosine receptor versus either the A1 or A2A receptors.
  • IB-MECA N 6 -(3-iodo-benzyl)-adenosine-5′-N-methyl-uronamide
  • the A3 adenosine receptor agonist may in particular be an N 6 -substituted-adenosine-5′-uronamide, such as an N 6 -monosubstituted-adenosine-5′-uronamide or an N 6 -disubstituted-adenosine-5′-uronamide.
  • the A3 adenosine receptor agonist may be one categorized as an N 6 -benzyladenosine-5′-uronamide; an N 6 -4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide; a 2-chloro-N 6 -substituted-4′-thioadenosine-5′-uronamide; or an (N)-methanocarba adenosine-5′-uronamide. Derivatives and analogs of compounds in these various classes may also be used.
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA); 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA); N 6 -(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (AB-MECA); N 6 -2-(4-aminophenyl) ethyladenosine (APNEA); 2-(1-hexynylhexynyl)-N-methyladenosine; 2-chloro-N 6 -(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]
  • Some other agonistic compounds include (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methyl amide (CP-532,903); and ring-constrained (N)-methanocarba-5′-uronamide 2,N 6 -disubstituted adenine nucleosides such as (1′R,2′R,3′S,4′R,5′S)-4- ⁇ 2-chloro-6-[(3-chlorophenylmethyl)amino]purin-9-yl ⁇ -1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol (MRS-3558; Tchilibon et al., 2005, J.
  • the A3 adenosine receptor agonist is selected from the group consisting of N 6 -(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA); 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-(N-methyluronamide) (Cl-IB-MECA); 2-(1-hexynylhexynyl)-N-methyladenosine; 2-chloro-N 6 -(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine (CF-102); and (2S,3S,4R,5R)-3-amino-5-[6-2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methyl amide (IB-MECA
  • Selective A3 adenosine receptor agonists include thionucleoside analogs of IB-MECA, such as those described in WO 2004/038006 and U.S. Pat. No. 7,199,127, including for example (2R,3S,4R)-2-[2-chloro-6-(3-iodobenzylamino)purin-9-yl]-3,4-dihydroxytetrahydrothiophene-2-carboxylic acid methyl amide; (2S,3S,4R,5R)-5-(2-chloro-6-methylaminopurin-9-yl)-3,4-dihydroxytetrahydrothiophene-2-carboxylic acid methyl amide; and (2S,3S,4R,5R)[5-(2-chloro-6-(3-iodobenzylamino)purin-9-yl)-3,4-dihydroxytetrahydrothiophene-2-carboxylic acid methyl
  • Selective A3 adenosine receptor agonists further include thioadenosine analogs of Cl-IB-MECA, as disclosed for example in Jeong et al., J Med Chem 2003:46:3775-3777; Jeong et al., J Med Chem 2006: 49:273-281; and Choi et al., Bioorg Med Chem 2009 Dec. 1; 17(23):8003-11.
  • A3 adenosine receptor agonists comprising a bicyclic ring substituent are described in U.S. Pat. No. 7,414,036, and include for example, N 6 -(4-iodo-2-picolyl)-adenosine-5′-N-methyluronamide; N 6 -(4-methyl-2-picolyl)-adenosine-5′-N-methyluronamide; N 6 -(2-picolyl)-adenosine-5′-N-methyluronamide; N 6 -(6-acetyl-2-picolyl)-adenosine-5′-N-methyluronamide; N 6 -(4-iodo-2-picolyl)-2-(2-phenyl-1-ethynyl)-adenosine-5′-N-methyluronamide; and N 6 -[(2-dimethylamino-7-iodo-1,3-benzoxazol-5-yl)
  • N 6 -4-sulfonamido-adenosine-5′-uronamide derivatives which are selective A3 adenosine receptor agonists are disclosed in U.S. Pat. No. 7,511,133 and in Baraldi et al., J. Med. Chem. 2004 Oct.
  • A3 adenosine receptor agonists may be used in the invention, such as those disclosed for example in U.S. Pat. Nos. 5,688,774; 5,773,423; 5,573,772; 5,443,836; 6,048,865; 6,177,444; 6,194,449; 6,323,214; 7,199,127; 7,414,036; 7,465,715; 7,511,133; U.S. Patent Application No. 2005/0250729; WO 95/02604; WO 99/20284; WO 99/06053; WO 97/27173; WO 01/23399; WO 02/055085; WO 02/070532; and WO 2004/038006.
  • an A3 adenosine receptor antagonist is any compound capable of specifically binding to the A3 adenosine receptor (A3R), and capable of fully or partially inhibiting i.e. inactivating said receptor.
  • the A3 adenosine receptor antagonist is thus a compound that exerts its prime effect though the binding and inactivation of the A3R.
  • Binding and functional assays that may be used for the characterization of such antagonists are essentially those that are used for the characterization of A3 adenosine receptor agonists, as described herein.
  • the A3 adenosine receptor antagonist has a binding affinity (Ki) for the A3 adenosine receptor that is less than about 200 nM, preferably less than about 100 nM, more preferably less than about 50 nM, even more preferably less than 10 nM.
  • the binding affinity of the A3 adenosine receptor antagonist for the adenosine A3 receptor is at least 20 times greater than the binding affinity of said antagonist for the A1 adenosine receptor.
  • A3 adenosine receptor antagonists which are useful in the present invention include a variety of known A3 adenosine receptor antagonists selected from but not limited to compounds variously classified as xanthines and derivatives thereof (as disclosed for example in Priego et al., J Med Chem. 2002 Aug. 1; 45(16):3337-44; and Muller et al., J Med Chem. 2002 Aug. 1; 45(16):3440-50; dihydropyridines and derivatives thereof (as disclosed for example in Jiang et al., 1997, J. Med. Chem. 40:2596-608 and U.S. Pat. No.
  • A3 adenosine receptor antagonists include 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523); 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334); 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-( ⁇ )-dihydropyridine-3,5 dicarboxylate (MRS-1191); 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-( ⁇ )-dihydropyridine-3,5-dicarboxylate (MRS-1097); 5-n-butyl-8-(4-trifluoromethylphenyl)
  • the A3 adenosine receptor antagonist is selected from the group consisting of 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523); 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334); and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-( ⁇ )-dihydropyridine-3,5 dicarboxylate (MRS-1191).
  • Suitable antagonists further include those disclosed in Jacobson et al., 1997, Neuropharmacology, 36:1157-65; Yao et al., 1997, Biochem. Biophys. Res. Commun., 232:317-22; Kim et al., 1996, J. Med. Chem., 39:4142-48; van Rhee et al., 1996, Drug Devel. Res., 37:131; van Rhee et al., 1996, J. Med. Chem., 39:2980-9; Siquidi et al., 1996, Nucleosides, Nucleotides 15:693-718; van Rhee et al., 1996, J. Med.
  • compositions and methods of the invention for increasing melanin production may further comprise the use of an allosteric enhancer of the A3 adenosine receptor.
  • an allosteric enhancer of the A3 adenosine receptor is disclosed for example in Gao et al., Mol Pharmacol. 2002 July; 62(1):81-9; Gao et al., Mol Pharmacol. 2001 November; 60(5):1057-63; and Goblyos et al., J Med Chem. 2006 Jun. 1; 49(11):3354-61.
  • allosteric enhancers of the A3 adenosine receptor include, but are not limited to, 1H-imidazo[4,5-c]quinolin-4-amines, 3-(2-pyridinyl)isoquinolines and 2,4-disubstituted quinolones.
  • 1H-imidazo[4,5-c]quinolin-4-amine include N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (LUF-6000); N-phenyl-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine (DU124183).
  • the 3-(2-pyridinyl)isoquinoline may be selected from 4-methoxy-N-[7-methyl-3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF5455); 4-methyl-N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF8502); 4-methoxy-N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF8504); and N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF8507).
  • the 2,4-disubstituted quinoline may be selected from N-(2-anilinoquinolin-4-yl)cyclopentanecarboxamide; N- ⁇ 2-[(3,4-dichlorophenyl)amino]quinolin-4-yl ⁇ cyclopentanecarboxamide; N-[2-(benzylamino)quinolin-4-yl]cyclopentanecarboxamide; N- ⁇ 2-[(4-methylphenyl)amino]quinolin-4-yl ⁇ cyclopentanecarboxamide; N-[2-(2,3-dihydro-1H-inden-5-ylamino)quinolin-4-yl]cyclopentanecarboxamide; N- ⁇ 2-[(4-methoxyphenyl)amino]quinolin-4-yl ⁇ cyclopentanecarboxamide; N- ⁇ 2-[(4-chlorophenyl)amino]quinolin-4-yl ⁇ cyclopentanecarbox
  • the skin lightening compositions of the invention may further contain at least one other adjunct ingredient in addition to the specific A3 adenosine receptor antagonist.
  • Adjunct ingredients include, but are not limited to, hydroxytetronic acid and/or hydroxytetronic acid derivatives, tetronic acid and/or tetronic acid derivatives, hydroquinone, ⁇ -hydroxy acids, and fatty acid esters of ascorbic acid. According to some embodiments some whitening compositions of the invention employ more than one adjunct ingredient.
  • Especially preferred whitening compositions of the invention that contain an adjunct ingredient employ either a hydroxytetronic acid and/or hydroxytetronic acid derivatives as those described in U.S. Pat. No. 7,019,029 or tetronic acid (2,4-furandione, formula C 4 H 6 O 5 ), or a tetronic acid derivative, or hydroquinone (sometimes also called p-dihydroxybenzene or 1,4 benzenediol), or both, in addition to the hydroxytetronic active ingredient in the formulation.
  • Tetronic acid and its derivatives have been suggested to be useful in the suppression of melanin production by inhibiting tyrosinase and tyrosine phosphatases (Sodeoka, M., 2001, J. Med.
  • tetronic acid adjunct ingredients are tetronic acid derivatives that inhibit tyrosinase or tyrosine phosphatase. These typically have an acyl or other hydrophobic group in the 3-position and a free tetronic acid moiety such as 5-(4-benzoylbenzoyl)oxymethyl-3-hexadecanoyltetronic acid reported by Sodeoka, et al., cited above.
  • Typical tetronic acid or tetronic acid derivative and/or hydroquinone concentrations range from about 0.25% to about 25% by weight, more narrowly from about 1% to about 5%, and even more narrowly from about 2% to about 4% by weight.
  • ⁇ -hydroxy acid has reference to and encompasses the general class of organic compounds containing at least one hydroxy group and at least one carboxyl group, and wherein at least one hydroxyl group is located on the ⁇ -carbon atom.
  • the compounds are organic acids having at least one carboxylic acid group and at least one hydroxyl group on the ⁇ -carbon atom, and may contain other functional groups including additional hydroxyl and carboxylic acid moieties.
  • Preferred ⁇ -hydroxy acids and/or ⁇ -hydroxy acid derivatives are those which are less bulky structurally, typically having a one- to three-carbon backbone, so that they penetrate the skin well such as those disclosed in U.S. Pat. No. 5,965,618.
  • glycolic and/or lactic acid or their derivatives are preferred.
  • Lactic acid was suggested as a skin-whitening agent in U.S. Pat. No. 5,262,153.
  • Typical hydroxy acid concentrations range from about 1% to about 25% by weight, more specifically from about 2% to about 15%, and even more specifically from about 3% to 10% by weight.
  • Typical hydroxytetronic acid concentrations range from about 8% to 12% by weight; more specifically from about 3% to about 7% by weight.
  • Fat-soluble fatty acid esters of ascorbic acid are employed as alternate or additional adjunct ingredients in other embodiments, alone or in combination with hydroquinone or ⁇ -hydroxy acids.
  • the more oxidation-resistant saturated fatty acid esters of ascorbic acid are preferred, including, but not limited to, ascorbyl laurate, ascorbyl myristate, ascorbyl palmitate, ascorbyl stearate, and ascorbyl behenate.
  • Ascorbyl palmitate is used in one embodiment.
  • the esters may be prepared using hydrogenated oils or fats, or fractions thereof. Ascorbyl stearate prepared using canola oil, for example, commonly contain about 4% ascorbyl palmitate.
  • fatty acid esters of ascorbic acid are employed as an adjunct ingredient, they help provide emollient properties to the composition.
  • Typical concentration ranges of ascorbyl palmitate vary from about 0.25% to about 10%, more narrowly from about 2% to about 8%, and even more narrowly from about 3% to about 5% by weight.
  • the invention encompasses therapeutic uses of A3 adenosine receptor antagonists for treating and ameliorating skin hyper-pigmentation disorders and conditions, as well as therapeutic uses of A3 adenosine receptor agonists for treating and ameliorating skin hypo-pigmentation disorders and conditions.
  • Hyper-pigmentation conditions which may be treated with the invention include for example, pigmented spots, lentigo senilis, freckles, café au lait spots, liver spots, ephelides, periorbital darkening, post-inflammatory hyper-pigmentation, pigmented keratosis, melasma and chloasma.
  • Hypo-pigmentation conditions which may be treated with the invention include for example, pigmented vitiligo, piebaldism, leukoderma due to cicatrisation, and nevus depigmentosis.
  • a preferred subject to be treated is a human.
  • A3 adenosine receptor antagonists for lightening dark skin, for example in individuals of population groups having genetically dark skin. Further encompassed within the invention are cosmetic uses of A3 adenosine receptor agonists for producing a tanned skin effect in individuals having fair skin.
  • A3 adenosine receptor agonists for treating depigmentation of a skin site which occurs secondary to surgical skin graft procedures
  • A3 adenosine receptor antagonists for treating hyperpigmentation of a skin site which occurs secondary to surgical skin graft procedures.
  • skin graft procedures may result in depigmentation or hyper-pigmentation at the graft site. Such an undesirable outcome may occur following cosmetic surgery, burn treatment, in particular split-thickness grafts or any other medical intervention requiring a skin graft procedure.
  • compositions comprising as and active ingredient an A3 adenosine receptor agonist or an A3 adenosine receptor antagonist may be administered to a subject by any route, includeing but are not limited to, direct administration into, onto or in the vicinity of hyper-pigmented or hypo-pigmented tissue, topical, intradermal, transdermal, subcutaneous, parenteral (including intravenous, intraarterial, intramuscular, intraperitoneal administration), as well as intranasal and oral administration.
  • the compounds can be administered by any convenient route and can be administered together with other therapeutically active agents, such as an allosteric enhancer of the A3 adenosine receptor or an adjunct skin lightening or whitening agent as described herein.
  • the route of administration is a route which enables the active ingredient i.e. A3 adenosine receptor agonist or A3 adenosine receptor antagonist to reach the blood stream, suitably intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or oral administration.
  • compositions of the invention may be administered locally to the hyper-pigmented or hypo-pigmented tissue in need of treatment; this can be achieved by, for example, and not by way of limitation, local injection or topical application, e.g., in conjunction with a wound dressing.
  • an effective amount of an A3 adenosine receptor agonist is that which results in a measurable or detectable increase in melanin level or skin pigmentation or relevant change in melanin location within or among cells.
  • an effective amount of an A3 adenosine receptor antagonist is that which results in a measurable or detectable decrease in melanin level or skin pigmentation or relevant change in melanin location within or among cells.
  • an effective amount is a daily administration of an A3 adenosine receptor agonist within the range of between about 1 ⁇ g/kg body weight and about 10 mg/kg body weight.
  • Such an amount of an A3 adenosine receptor agonist is typically administered in a single daily dose although at times a daily dose may be divided into several doses administered throughout the day or at times several daily doses may be combined into a single dose to be given to the patient once every several days, particularly if administered in a sustained or controlled release formulation.
  • Administration of the A3 adenosine receptor agonist or the A3 adenosine receptor may be carried out over a period of at least two weeks, or at least one month, or at least two months, or longer, in order to achieve the desired outcome.
  • compositions of the present invention may contain, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can performs its intended function.
  • a carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • compositions of the invention can be applied to the skin. According to some other embodiments, the compositions of the invention can be applied to hair follicles.
  • the compositions may be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, pad or gelled stick.
  • A3 adenosine receptor agonists and antagonists can be delivered in a controlled release system.
  • the A3 Adenosine receptor agonists and antagonists of the invention can be administered in combination with a biodegradable, biocompatible polymeric implant, which releases the A3 Adenosine receptor antagonist or agonist over a controlled period of time at the hyper-pigmented or hypo-pigmented tissue respectively.
  • a biodegradable, biocompatible polymeric implant which releases the A3 Adenosine receptor antagonist or agonist over a controlled period of time at the hyper-pigmented or hypo-pigmented tissue respectively.
  • preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (see Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.).
  • the A3 adenosine receptor agonist or antagonist is administered as part of a topical formulation.
  • the compositions of the present invention formulated for topical administration comprise the melanin modulating agent in an effective dose together with one or more pharmaceutically or therapeutically acceptable carriers and optionally other ingredients.
  • a pharmaceutically or therapeutically acceptable carriers and optionally other ingredients.
  • Various considerations are described, e.g., in Gilman et al. (eds) (1990) The Pharmacological Bases Of Therapeutics, 8th Ed., Pergamon Press; Novel Drug Delivery Systems, 2nd Ed., Norris (ed.) Marcel Dekker Inc. (1989), and Remington's Pharmaceutical Sciences.
  • the formulations will comprise a preparation for delivering a melanin modulating agent directly to the skin or hair comprising the modulating agent, typically in concentrations in the range from about 0.001% to 20%; preferably, from about 0.01 to about 10%; more preferably, from about 0.1 to about 5%; together with a non-toxic, pharmaceutically acceptable topical carrier.
  • Topical preparations can be prepared by combining the melanin modulating agent with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream and aerosol formulations.
  • suitable dosage forms e.g., sprays, ointments, pastes, creams, lotions, gels, and solutions
  • suitable dosage forms will comprise mixing the active compound under sterile conditions with diluents and carriers along with any preservatives, buffers, or propellants which may be required.
  • Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • bases may include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil.
  • Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
  • Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like.
  • Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like.
  • the topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like.
  • the topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
  • active and/or adjunct ingredients are added to a sunscreen or sunblock formulations so that topical application has the further advantage of preventing repigmentation during and/or after treatment.
  • Preferred formulae of this type are SPF 15 or higher.
  • Many of these preferred embodiments contain titanium dioxide or zinc oxide which additionally smooth and lubricate the skin and help minimize side effects in sensitive skin.
  • Parenterally administered formulations are generally prepared in a unit dosage injectable form (solution, suspension, emulsion).
  • a pharmaceutical formulation suitable for injection includes sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier employed can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, lipid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils.
  • Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and ester, such as isopropyl myristate, may also at times be used as solvent systems for the active ingredient.
  • compositions which enhance the stability, sterility and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers can be added. Prevention of microbial growth can be ensured by inclusion of various preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid and the like.
  • the active ingredient may be formulated in the form of tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices
  • compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • opacifying agents include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • ameliorate is used here in its broadest sense to refer to the reduction of the pigmentation in patients suffering from hyper-pigmentation. Alternatively, it refers to an increase in pigmentation in patient suffering from hypo-pigmentation. The term includes any of the arrest, prevention, decrease, and improvement in any of the symptoms of hyper-pigmentation and hypo-pigmentation, both temporary and long term. Amelioration of hyper-pigmentation or hypo-pigmentation is a further example of successful treatment or therapy.
  • IB-MECA, MRS-1523 and IBMX were purchased from Sigma.
  • DMSO dimethyl sulfoxide
  • DMEM medium Dulbecco's modified Eagle's medium
  • DMEM media, fetal calf serum, and antibiotics for cell cultures were obtained from Kibbutz Beit-Haemek, Israel.
  • Kojic acid, synthetic melanin and anti-phospho-ERK antibody were purchased from Sigma.
  • Anti-ERK-2 antibody and anti-phospho-ERK antibody were purchased from Santa Cruz Biotechnology.
  • Anti-Akt-1 antibody and anti-phospho-Akt antibody were purchased from Cell Signaling, and anti-Mitf antibody was purchased from Abcam.
  • BCATM Protein Assay Kit and ECL Kit were purchased from Thermo Scientific.
  • B16 F1 melanoma cells were grown adherently and maintained in DMEM (without phenol red), containing 10% fetal calf serum, 1% antibiotics (penicillin, streptomycin and nystatin, each at 100 U/ml), and 2 mM L-glutamine. Cells were cultured at 37° C. in 5% CO 2 , and passaged two to three times weekly at a ratio of 1:10.
  • the resolved proteins were then electroblotted onto nitrocellulose membrane.
  • Membranes were blocked with 1% bovine serum albumin and incubated with anti-ERK-2 antibody (1:1000) or anti-phospho-ERK antibody (1:10000) for 1 h at room temperature, or with anti-Akt-1 antibody (1:1000) or with anti-phospho-Akt antibody (1:000) at 4° C. for 24 h. Blots were then washed and incubated with horseradish peroxidase (conjugated secondary antibodies), for 1 hour at room temperature. The proteins were detected using ECL kit. For the detection of ERK phosphorylation after 72 hours of exposure to the ligands, non-starved cells were used (with 10% FCS).
  • B16 melanocytes were grown for 24 h on glass cover-slips coated with poly-L-lysine (500 ug/ml) in 6 wells plates in the presence of IB-MECA or MRS-1523 (10 ⁇ M), for 3 h or 24 h at 37° C.
  • Cells were fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 1 h at room temperature. The fixed cells were rinsed three times for 1 min with PBS. To block non-specific interaction of the antibodies, cells were incubated with PBS (1% BSA, 0.1% Triton X-100).
  • cells were incubated with the primary antibody at a dilution of 1:1000 for 24 h at 4° C. Cells were washed three times for 3 min with PBS and incubated with Cy3-conjugated donkey anti-rabbit antibody at a dilution of 1:250 in PBS for 2 h in the dark. Cells were washed three times with PBS and mounted with Fluoromount (Sigma). Cells were visualized by confocal microscopy (excitation 553 and emission at 568, Lieca).
  • cells were treated as described above and incubated with primary anti-Mitf antibody at a dilution of 1:100 for 24 h at 4° C. and with the secondary antibody Alexa Fluor® 488-conjugated donkey anti-mouse antibody at a dilution of 1:250 for 2 h in the dark.
  • Skin organ culture Samples of human breast skin obtained during breast surgery were cut into pieces (0.5 ⁇ 0.5 cm) and cultured in DMEM medium (described above) in 6 well plates and incubated at 37° C. with 5% CO 2 for 12 days. Skin samples were topically treated with 10 or 50 ⁇ M of various A3 adenosine receptor ligands or 100 ⁇ M hydroquinone three times during the incubation period and the medium was exchanged every 3 days.
  • DOPA staining Treated skin samples were washed with PBS and the epidermal sheets were separated from the dermis and incubated in 0.1% L-3,4-DOPA in PBS for 4 h at 37° C. The epidermis sheets were then fixed with 4% formaldehyde solution, dehydrated by graded alcohol, cleared in xylene, and mounted on glass slides.
  • DOPA positive melanocytes were visualized by light microscopy ( ⁇ 400 magnification).
  • Light transmission capacity of the epidermis sheet was determined by taking 3 images of each sheet from different zones of the epidermis of each treatment ( ⁇ 50 magnification). Light transmission of each area in the epidermis sheet was calculated and expressed as a mean of density of three images.
  • MRS-1523 affected both the synthesis and the secretion of melanin by the cells as shown in FIG. 2 .
  • Exposure of melanocytes to 40 ⁇ M MRS-1523 caused a decrease in total melanin by 67 ⁇ 1.07% (p ⁇ 0.001; FIG. 2B ).
  • This treatment effectively blocked melanin secretion by 88 ⁇ 1.7%, while the melanin level within the cells decreased only by 11 ⁇ 2.3% ( FIG. 2A ).
  • the melanin level in the cells decreased only by 27 ⁇ 5.7% and 24 ⁇ 1.7% respectively.
  • this treatment was associated with a significant decrease of the intracellular melanin (48% ⁇ 4.9, p ⁇ 0.05).
  • the secreted and the total melanin levels were lower than that of the control (p ⁇ 0.05, FIGS. 2A and 2B ).
  • B16 melanocytes were exposed to 10 ⁇ M of IB-MECA or 10 ⁇ M MRS-1523 and the melanin levels in the cells and in the media were monitored at different time points following exposure to the ligands.
  • confocal microscopy showed observable changes in melanin level and distribution in the cytosol of the treated cells.
  • FIG. 3B depicts transfer of melanosomes from one melanocyte to the other by a dendrite-dendrite interaction. Moreover the melanosomes appear to be injected and moved along the edge of cells.
  • FIG. 3C shows total melanin levels after various intervals of exposure to IB-MECA or MRS-1523.
  • the level of total melanin was non-significantly increased.
  • the total melanin level i.e. melanin in the cells and melanin secreted to the media
  • the total melanin level was significantly increased by 198 ⁇ 12.1% (p ⁇ 0.05).
  • the total melanin level was significantly decreased by 67 ⁇ 4.4% (p ⁇ 0.05).
  • FIG. 3E is a photograph showing melanin secretion to the media in control cells and in cells exposed to IB-MECA or MRS-1523 for 5 days. Consistent with the results shown in FIGS. 3C and 3D , prolonged exposure of melanocytes to IB-MECA resulted in a visually observable increase in melanin, whereas prolonged exposure of cells to MRS-1523 resulted in a visually observable decrease in melanin.
  • Kojic acid is a well known melanization decreasing agent.
  • the melanin synthesis in cells has been well studied; however, no information regarding the effect of kojic acid on melanin secretion, which constitutes a major contribution to skin melanization, was obtained.
  • B16 melanocytes were cultured in 10% DMEM in the presence of different concentrations of kojic acid for 5 days. The melanin and protein content were measured as described in Materials and Methods.
  • FIG. 4B kojic acid exerted inhibition of 51% ⁇ 2.4 in the total melanin level (p ⁇ 0.001).
  • FIG. 4A shows that both melanin synthesis (overall melanin levels) and secretion (melanin level in the medium) were affected.
  • FIG. 5A The results shown in FIG. 5A indicate that both the A3 adenosine receptor agonist (IB-MECA) and the A3 adenosine receptor antagonist (MRS-1523) induced phosphorylation of Akt within 10 min. However, continued exposure to MRS-1523 (up to 3 h) led to downregulation of P-Akt ( FIG. 5A left panel), while continued exposure to IB-MECA resulted in increased phosphorylation of Akt ( FIG. 5A right panel).
  • IB-MECA A3 adenosine receptor agonist
  • MRS-1523 A3 adenosine receptor antagonist
  • Akt family signal pathway has been shown to be upstream to the ERK signal pathway, and the increase in phosphorylated Akt is implicated in downregulation of phosphorylated ERK. Without wishing to be bound by any particular mechanism and theory, it is contemplated that increasing the level of phosphorylated ERK leads to downregulation of the transcription factor Mitf which regulates tyrosinase, a key enzyme in melanogenesis and cell differentiation.
  • Mitf which regulates tyrosinase
  • ERK signaling pathway has been shown to be upstream to the transcription factor Mitf and downstream to the Akt signaling pathway.
  • ERK phosphorylation analyses show that MRS-1523 induced phophorylation of ERK 1 within 30 minutes and phosphorylation of ERK 2 within 10 minutes ( FIG. 6A ).
  • IB-MECA (10 ⁇ M) did not increase the level of ERK phosphorylation ( FIG. 6A ).
  • Mitf is a transcription factor which regulates tyrosinase, a key enzyme in melanogenesis and cell differentiation and has been shown to be downstream to ERK.
  • FIG. 7 shows that Mitf is highly expressed in the control and IB-MECA treated cells ( FIG. 7 , left and middle panels).
  • IB-MECA A3 adenosine receptor agonist
  • FIG. 7 shows that Mitf is highly expressed in the control and IB-MECA treated cells ( FIG. 7 , left and middle panels).
  • exposure of cells to the A3 adenosine receptor agonist IB-MECA induced translocation of Mitf protein to the nucleus within 3 h of exposure, while in control cells Mitf remained in the cytosol.
  • the expression of Mitf in IB-MECA treated cells remained high, both in the cytoplasm and in the nucleus.
  • FIGS. 8A-8E indicate that topical treatment of human skin explants with the A3 adenosine receptor antagonist MRS-1523 decreased melanin levels, whereas treatment with the A3 adenosine receptor agonist IB-MECA increased melanin levels, both after 12 days of exposure. It was further observed that hydroquinone, usually recognized as a whitening agent, increased skin pigmentation when applied topically at a concentration of 100 ⁇ M for 12 days.
  • FIGS. 8B and 8D show the increased number of dark-stained DOPA positive melanocytes (D-P-M) in IB-MECA-treated and hydroquinone-treated samples as compared to control.
  • FIGS. 8B and 8D further show the decreased number of D-P-M in MRS-1523-treated samples as compared to control.
  • the histological appearance of IB-MECA-treated samples suggests that the observed increase in skin pigmentation is due to an elevation in melanin accumulation and/or deposition in the adjacent keratinocytes, rather than an increase in the number of melanocytes (see FIG. 9A ).
  • the melanocytes in MRS-1523-treated cells appear to contain less melanin and there is a lesser extent of melanin accumulation and/or deposition in the keratinocytes (see FIG. 9D ).

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